Evaluation of a modified rapid viability-polymerase chain reaction method for Bacillus atrophaeus spores in water matrices.

A rapid method that provides information on the viability of organisms is needed to protect public health and ensure that remediation efforts following a release of a biological agent are effective. The rapid viability-polymerase chain reaction (RV-PCR) method combines broth culture and molecular methods to provide results on whether viable organisms are present in less than 15 h. In this study, a modified RV-PCR (mRV-PCR) method was compared to a membrane-filtration culture method for the detection of viable Bacillus spores in water matrices. Samples included small and large volumes of chlorine and non­chlorine treated tap water. Large volume water samples (up to 100 L), were processed by ultrafiltration using a semi-automated waterborne pathogen concentrator, followed by centrifugation as a secondary concentration technique. The concentrated samples were analyzed by mRV-PCR and culture methods. The overall agreement between the mRV-PCR and culture methods when seed concentrations were greater than 10 spores per sample volume analyzed was 96%. The total time from the start of sample processing to the final sample result for the mRV-PCR method was decreased by approximately 2 h, in comparison to the previously published RV-PCR method because of the incorporation of shorter, more efficient primary and secondary concentration steps and a shorter DNA extraction technique. Overall, this study confirmed that RV-PCR is a promising approach for identifying viable Bacillus spores in small- and large-volume water samples and for producing results in less time than traditional culture methods.

Predicting microcystin concentration action-level exceedances resulting from cyanobacterial blooms in selected lake sites in Ohio.

Cyanobacterial harmful algal blooms and the toxins they produce are a global water-quality problem. Monitoring and prediction tools are needed to quickly predict cyanotoxin action-level exceedances in recreational and drinking waters used by the public. To address this need, data were collected at eight locations in Ohio, USA, to identify factors significantly related to observed concentrations of microcystins (a freshwater cyanotoxin) that could be used in two types of site-specific regression models. Real-time models include easily or continuously-measured factors that do not require that a sample be collected; comprehensive models use a combination of discrete sample-based measurements and real-time factors. The study sites included two recreational sites and six water treatment plant sites. Real-time models commonly included variables such as phycocyanin, pH, specific conductance, and streamflow or gage height. Many real-time factors were averages over time periods antecedent to the time the microcystin sample was collected, including water-quality data compiled from continuous monitors. Comprehensive models were useful at some sites with lagged variables for cyanobacterial toxin genes, dissolved nutrients, and (or) nitrogen to phosphorus ratios. Because models can be used for management decisions, important measures of model performance were sensitivity, specificity, and accuracy of estimates above or below the microcystin concentration threshold standard or action level. Sensitivity is how well the predictive tool correctly predicts exceedance of a threshold, an important measure for water-resource managers. Sensitivities > 90% at four Lake Erie water treatment plants indicated that models with continuous monitor data were especially promising. The planned next steps are to collect more data to build larger site-specific datasets and validate models before they can be used for management decisions.

Multi-Year Microbial Source Tracking Study Characterizing Fecal Contamination in an Urban Watershed.

Microbiological and hydrological data were used to rank tributary stream contributions of bacteria to the Little Blue River in Independence, Missouri. Concentrations, loadings and yields of E. coli and microbial source tracking (MST) markers, were characterized during base flow and storm events in five subbasins within Independence, as well as sources entering and leaving the city through the river. The E. coli water quality threshold was exceeded in 29% of base-flow and 89% of storm-event samples. The total contribution of E. coli and MST markers from tributaries within Independence to the Little Blue River, regardless of streamflow, did not significantly increase the median concentrations leaving the city. Daily loads and yields of E. coli and MST markers were used to rank the subbasins according to their contribution of each constituent to the river. The ranking methodology used in this study may prove useful in prioritizing remediation in the different subbasins.

Estimating microcystin levels at recreational sites in western Lake Erie and Ohio.

Cyanobacterial harmful algal blooms (cyanoHABs) and associated toxins, such as microcystin, are a major global water-quality issue. Water-resource managers need tools to quickly predict when and where toxin-producing cyanoHABs will occur. This could be done by using site-specific models that estimate the potential for elevated toxin concentrations that cause public health concerns. With this study, samples were collected at three Ohio lakes to identify environmental and water-quality factors to develop linear-regression models to estimate microcystin levels. Measures of the algal community (phycocyanin, cyanobacterial biovolume, and cyanobacterial gene concentrations) and pH were most strongly correlated with microcystin concentrations. Cyanobacterial genes were quantified for general cyanobacteria, general Microcystis and Dolichospermum, and for microcystin synthetase (mcyE) for Microcystis, Dolichospermum, and Planktothrix. For phycocyanin, the relations were different between sites and were different between hand-held measurements on-site and nearby continuous monitor measurements for the same site. Continuous measurements of parameters such as phycocyanin, pH, and temperature over multiple days showed the highest correlations to microcystin concentrations. The development of models with high R2 values (0.81-0.90), sensitivities (92%), and specificities (100%) for estimating microcystin concentrations above or below the Ohio Recreational Public Health Advisory level of 6µgL-1 was demonstrated for one site; these statistics may change as more data are collected in subsequent years. This study showed that models could be developed for estimates of exceeding a microcystin threshold concentration at a recreational freshwater lake site, with potential to expand their use to provide relevant public health information to water resource managers and the public for both recreational and drinking waters.

Holding-time and method comparisons for the analysis of fecal-indicator bacteria in groundwater.

As part of the US Geological Survey National Water-Quality Assessment Program, groundwater samples from domestic- and public-supply wells were collected and analyzed for fecal-indicator bacteria. A holding time comparison for total coliforms, Escherichia coli, and enterococci was done by analyzing samples within 8 h using presence/absence methods and within 18-30 h using quantitative methods. The data indicate that results obtained within 18-30 h were not significantly different from those obtained within 8 h for total coliforms and enterococci, by Colilert® and Enterolert® methods (IDEXX Laboratories Inc., Westbrook, ME), respectively. Quantitative laboratory methods for samples analyzed within 18-30 h showed a statistically significant higher detection frequency when compared to presence/absence methods done within 8 h for the following methods, E. coli by Colilert and enterococci by membrane filtration on mEI agar. Additionally, a comparison of methods for the enumeration of enterococci was done. Using non-parametric statistical analyses, results from the two methods were statistically different. In this study, the membrane filtration method on mEI agar was more sensitive, resulted in more detections of enterococci, and results were easier to interpret than with the quantitative Enterolert method. The quantitative Enterolert method produced varying levels of fluorescence, which required additional verification steps to eliminate false-positive results. It may be more advantageous to analyze untreated groundwater for enterococci using the membrane filtration method on mEI agar.

Predictive models for Escherichia coli concentrations at inland lake beaches and relationship of model variables to pathogen detection.

Predictive models, based on environmental and water quality variables, have been used to improve the timeliness and accuracy of recreational water quality assessments, but their effectiveness has not been studied in inland waters. Sampling at eight inland recreational lakes in Ohio was done in order to investigate using predictive models for Escherichia coli and to understand the links between E. coli concentrations, predictive variables, and pathogens. Based upon results from 21 beach sites, models were developed for 13 sites, and the most predictive variables were rainfall, wind direction and speed, turbidity, and water temperature. Models were not developed at sites where the E. coli standard was seldom exceeded. Models were validated at nine sites during an independent year. At three sites, the model resulted in increased correct responses, sensitivities, and specificities compared to use of the previous day's E. coli concentration (the current method). Drought conditions during the validation year precluded being able to adequately assess model performance at most of the other sites. Cryptosporidium, adenovirus, eaeA (E. coli), ipaH (Shigella), and spvC (Salmonella) were found in at least 20% of samples collected for pathogens at five sites. The presence or absence of the three bacterial genes was related to some of the model variables but was not consistently related to E. coli concentrations. Predictive models were not effective at all inland lake sites; however, their use at two lakes with high swimmer densities will provide better estimates of public health risk than current methods and will be a valuable resource for beach managers and the public.

Comparison of filters for concentrating microbial indicators and pathogens in lake water samples.

Bacterial indicators are used to indicate increased health risk from pathogens and to make beach closure and advisory decisions; however, beaches are seldom monitored for the pathogens themselves. Studies of sources and types of pathogens at beaches are needed to improve estimates of swimming-associated health risks. It would be advantageous and cost-effective, especially for studies conducted on a regional scale, to use a method that can simultaneously filter and concentrate all classes of pathogens from the large volumes of water needed to detect pathogens. In seven recovery experiments, stock cultures of viruses and protozoa were seeded into 10-liter lake water samples, and concentrations of naturally occurring bacterial indicators were used to determine recoveries. For the five filtration methods tested, the highest median recoveries were as follows: glass wool for adenovirus (4.7%); NanoCeram for enterovirus (14.5%) and MS2 coliphage (84%); continuous-flow centrifugation (CFC) plus Virocap (CFC+ViroCap) for Escherichia coli (68.3%) and Cryptosporidium (54%); automatic ultrafiltration (UF) for norovirus GII (2.4%); and dead-end UF for Enterococcus faecalis (80.5%), avian influenza virus (0.02%), and Giardia (57%). In evaluating filter performance in terms of both recovery and variability, the automatic UF resulted in the highest recovery while maintaining low variability for all nine microorganisms. The automatic UF was used to demonstrate that filtration can be scaled up to field deployment and the collection of 200-liter lake water samples.

Comparative effectiveness of membrane bioreactors, conventional secondary treatment, and chlorine and UV disinfection to remove microorganisms from municipal wastewaters.

Log removals of bacterial indicators, coliphage, and enteric viruses were studied in three membrane bioreactor (MBR) activated-sludge and two conventional secondary activated-sludge municipal wastewater treatment plants during three recreational seasons (May-Oct.) when disinfection of effluents is required. In total, 73 regular samples were collected from key locations throughout treatment processes: post-preliminary, post-MBR, post-secondary, post-tertiary, and post-disinfection (UV or chlorine). Out of 19 post-preliminary samples, adenovirus by quantitative polymerase chain reaction (qPCR) was detected in all 19, enterovirus by quantitative reverse transcription polymerase chain reaction (qRT-PCR) was detected in 15, and norovirus GI by qRT-PCR was detected in 11. Norovirus GII and Hepatitis A virus were not detected in any samples, and rotavirus was detected in one sample but could not be quantified. Although culturable viruses were found in 12 out of 19 post-preliminary samples, they were not detected in any post-secondary, post-MBR, post-ultraviolet, or post-chlorine samples. Median log removals for all organisms were higher for MBR secondary treatment (3.02 to >6.73) than for conventional secondary (1.53-4.19) treatment. Ultraviolet disinfection after MBR treatment provided little additional log removal of any organism except for somatic coliphage (>2.18), whereas ultraviolet or chlorine disinfection after conventional secondary treatment provided significant log removals (above the analytical variability) of all bacterial indicators (1.18-3.89) and somatic and F-specific coliphage (0.71 and >2.98). Median log removals of adenovirus across disinfection were low in both MBR and conventional secondary plants (no removal detected and 0.24), and few removals of individual samples were near or above the analytical variability of 1.2 log genomic copies per liter. Based on qualitative examinations of plots showing reductions of organisms throughout treatment processes, somatic coliphage may best represent the removal of viruses across secondary treatment in both MBR and conventional secondary plants. F-specific coliphage and Escherichia coli may best represent the removal of viruses across the disinfection process in MBR facilities, but none of the indicators represented the removal of viruses across disinfection in conventional secondary plants.

Comparison of immunomagnetic separation/adenosine triphosphate rapid method to traditional culture-based method for E. coli and enterococci enumeration in wastewater.

Untreated wastewater samples from California, North Carolina, and Ohio were analyzed by the immunomagnetic separation/adenosine triphosphate (IMS/ATP) method and the traditional culture-based method for E. coli and enterococci concentrations. The IMS/ATP method concentrates target bacteria by immunomagnetic separation and then quantifies captured bacteria by measuring bioluminescence induced by release of ATP from the bacterial cells. Results from this method are available within 1h from the start of sample processing. Significant linear correlations were found between the IMS/ATP results and results from traditional culture-based methods for E. coli and enterococci enumeration for one location in California, two locations in North Carolina, and one location in Ohio (r values ranged from 0.87 to 0.97). No significant linear relation was found for a second location in California that treats a complex mixture of residential and industrial wastewater. With the exception of one location, IMS/ATP showed promise as a rapid method for the quantification of faecal-indicator organisms in wastewater.

Possible linkages between lignite aquifers, pathogenic microbes, and renal pelvic cancer in northwestern Louisiana, USA.

In May and September, 2002, 14 private residential drinking water wells, one dewatering well at a lignite mine, eight surface water sites, and lignite from an active coal mine were sampled in five Parishes of northwestern Louisiana, USA. Using a geographic information system (GIS), wells were selected that were likely to draw water that had been in contact with lignite; control wells were located in areas devoid of lignite deposits. Well water samples were analyzed for pH, conductivity, organic compounds, and nutrient and anion concentrations. All samples were further tested for presence of fungi (cultures maintained for up to 28 days and colonies counted and identified microscopically) and for metal and trace element concentration by inductively-coupled plasma mass spectrometry and atomic emission spectrometry. Surface water samples were tested for dissolved oxygen and presence of pathogenic leptospiral bacteria. The Spearman correlation method was used to assess the association between the endpoints for these field/laboratory analyses and incidence of cancer of the renal pelvis (RPC) based on data obtained from the Louisiana Tumor Registry for the five Parishes included in the study. Significant associations were revealed between the cancer rate and the presence in drinking water of organic compounds, the fungi Zygomycetes, the nutrients PO(4) and NH(3), and 13 chemical elements. Presence of human pathogenic leptospires was detected in four out of eight (50%) of the surface water sites sampled. The present study of a stable rural population examined possible linkages between aquifers containing chemically reactive lignite deposits, hydrologic conditions favorable to the leaching and transport of toxic organic compounds from the lignite into the groundwater, possible microbial contamination, and RPC risk.